PROJECT SUMMARY Most tumors go undetected until advanced stages when treatment is more expensive, less effective, and poses a greater risk for secondary effects on patient health. In this proposal, we focus on a fundamental, yet understudied aspect of cancer: the emergence and evolution of early tumors. To better understand the cellular and molecular basis of early tumor biology, we need to directly observe the process of malignant transformation, however, this is nearly impossible to do using in vivo models. To overcome these challenges, we propose to develop a suit of experimental and computational approaches that will allow us to study the emergence of tumors with unprecedented detail. Our approach relies on innovative ex vivo culture systems that we designed to mimic the physiological conditions of the cellular microenvironment. In these systems, we will monitor the development of malignant clones using high-throughput live microscopy, metabolomics, and single-cell genomics. In the current model of malignant transformation, normal cells accumulate mutations at random, some of which are oncogenic and lead to faster cell growth and additional cancer hallmarks. In this view, the environment plays a secondary role limited to the selection of oncogenic mutations and the elimination of detrimental ones. We will test this model against one where the environment triggers malignant-like behaviors that anticipate the acquisition of genetic malignancy. In this alternative model, phenotypically malignant cells are more likely to become genetically transformed cells because they are better adapted to challenging environments and have epigenetic modifications that facilitate the acquisition of oncogenic mutations. To test this hypothesis, we will perform long-term evolution experiments in which we will study the spontaneous emergence and evolution of malignant clones from populations growing under tightly-controlled and reproducible environments. We will track changes in these populations using deep sequencing and automated imaging over many cell generations. In parallel, we will use metabolomics and synthetic biology tools to measure and perturb the local environments in which different cells grow. Overall, this proposal will allow us to study the cellular, molecular, and environmental factors that affect the emergence of malignant cells and will help us understand how challenging microenvironments accelerate the evolution of malignant clones. Our long-term goal is to gain fundamental knowledge about the initiating steps of tumorigenesis, so that we can better understand ? and potentially treat ? early tumors. Preventing tumors at their earliest stages will bring us closer to a cure for cancer, and our research is an attempt to contribute towards this larger goal. With the expertise of my lab, our scientific environment, and the tools we have developed, I believe we are in an advantageous position to embark on this ambitious, but urgently needed research program.